Insulin Receptor Substrate-1 Protein Expression Research Articles

Intracerebroventricular Injection of MHY1485 Blocked the Beneficial Effect of Adiponectin on Aversive Memory in the STZ Model of Dementia.

The most prominent adipokine, adiponectin (APN), has an adverse relationship with the malfunction of adipose tissue. Obesity causes a decrease in plasma APN levels, which eventually results in insulin resistance and diabetes. In this study, we assessed how the effects of APN on memory are influenced by the insulin receptor substrate-1 (IRS-1) and the mammalian target of rapamycin (mTOR) pathways. Streptozotocin (STZ) 3 mg/kg intracerebroventricular injections on days 1 and 3 following cannulation were used to create an animal model of Alzheimer's disease. The acquisition phase was preceded by injections of MHY and adiponectin. For the passive avoidance task, the stepthrough latency and total duration in the dark compartment were recorded and evaluated, and the preference index was calculated for the novel object identification test. IRS-1 protein expression in the hippocampus was assessed by western blotting. STZ reduced the step-through latency (STL), which rose significantly (P≤0.001) in the APN+STZ group. The memory-improving effects of APN were reversed when MHY was administered first (P≤0.001). The STZ and APN+STZ+MHY groups both had a substantial decline in the preference index (P≤0.01). Compared to the control group, the STZ group's expression of the IRS- 1 protein was dramatically reduced (P≤0.0001). In contrast to the APN+STZ group, the MHYtreated group likewise showed decreased IRS-1 protein expression (P≤0.0001), but APN+STZ was able to enhance IRS-1 expression rate (P≤0.0001). In a rat model of AD, we found that adiponectin improved aversive and cognitive memory, which is at least partially mediated by the mTOR signaling cascade.

Correlation Study on the Expression of INSR, IRS-1, and PD-L1 in Nonsmall Cell Lung Cancer.

Objective To study the expression and correlation of insulin receptor (INSR), insulin receptor substrate-1 (IRS-1), and programmed cell death ligand-1 (PD-L1) in nonsmall cell lung cancer (NSCLC). Methods 45 lung cancer tissues and 30 adjacent normal tissues of NSCLC patients diagnosed in the Second Affiliated Hospital of Shandong First Medical University from June 2019 to August 2020 were selected. The expressions of INSR, IRS-1, and PD-L1 proteins in tumor tissues and adjacent tissues of NSCLC were detected by immunohistochemical staining. Results The expression of INSR and IRS-1 in NSCLC was significantly higher than that in adjacent normal lung tissue (P < 0.05). INSR expression had statistical significance with the degree of pathological differentiation of nonsmall cell carcinoma (P = 0.031), but had no significant association with age, gender, pathological type, TNM stage, and lymph node metastasis status (P > 0.05). There was no significant correlation between IRS-1 positive expression and NSCLC patients' age, gender, pathological typing, degree of differentiation, TNM stage, and lymph node metastasis (P > 0.05). PD-L1 positive expression was correlated with lymph node metastasis of NSCLC (P = 0.028), while there was no significant correlation with gender, age, pathological type, TNM stage, and pathological differentiation degree of NSCLC patients (P > 0.05). Spearman correlation analysis showed that PD-L1 protein expression had a significant positive correlation with IRS-1 protein expression (r = 0.373), but was not correlated with the expression of INSR protein. Conclusion IRS-1 may be involved in the regulation of PD-L1 expression and mediate the occurrence of tumor immune escape, which is expected to become a new target for NSCLC immunotherapy and provide new clinical evidence for immunosuppressive therapy.

The miRNA-144-5p/IRS1/AKT axis regulates the migration, proliferation, and mineralization of osteoblasts: A mechanism of bone repair in diabetic osteoporosis.

Diabetic osteoporosis (DOP) is a disorder of bone metabolism induced by multiple mechanisms. Previous studies have revealed that microRNAs (miRNAs) play crucial roles in bone metabolism. MiRNA-144-5p has been proven to participate in the regulation of osteoblast activities; however, its specific mechanism in DOP has not been elucidated. This study investigated whether high glucose (HG) inhibited osteoblasts by regulating miRNA-144-5p. Our results showed that HG inhibited bone formation not only in vivo but also in vitro. We observed that HG severely hindered the migration, proliferation and mineralization of osteoblasts, while miRNA-144-5p was upregulated by way of the cell counting kit-8 assay, wound healing assay, alkaline phosphatase (ALP) activity assay and alizarin red staining. Double luciferase reporter experiments showed that miRNA-144-5p directly targeted insulin receptor substrate 1 (IRS1). The IRS1/AKT signaling pathway is closely related to osteoblasts' migration, proliferation, and mineralization. Silencing miRNA-144-5p promoted the mRNA, and protein expression of IRS1, thereby letting the expression of total AKT down, and then preventing phosphorylation of AKT into the nucleus to regulate migration, proliferation, and mineralization genes of osteoblasts. In conclusion, this study indicated that HG regulated the migration, proliferation, and mineralization of osteoblasts via the miRNA-144-5p/IRS1/AKT axis, which suggested a possible mechanism for DOP pathology.

Effect of electroacupuncture on protein expressions of SOCS3 and IRS-1 in hypothalamus and pancreas islet morphology in diabetic fatty rats

To observe the effect of electroacupuncture (EA) on protein expressions of suppressor of cytokine signaling 3 (SOCS3) and insulin receptor substrate-1 (IRS-1) in hypothalamus and morphology of pancreas islet in Zucker diabetic fatty (ZDF) rats, and to explore its possible mechanism on improving plasma glucose and insulin resistance of type 2 diabetes mellitus (T2DM). Twelve SPF male ZDF rats were selected and fed with high-fat diet for 4 weeks to establish the T2DM model, after modeling, the rats were randomly divided into a model group and an EA group, 6 rats in each one. Besides, 6 SPF male Zucker lean rats were selected as a blank group. In the EA group, EA was applied at "Pishu" (BL 20), "Weiwanxiashu" (EX-B 3), "Zusanli" (ST 36) and "Sanyinjiao" (SP 6), with continuous wave, 15 Hz in frequency, 2 mA in intensity, once a day, 20 min each time, 6 times a week for 4 weeks. The fasting plasma glucose (FPG) was measured before and after intervention. The serum level of fasting insulin (FINS) was measured by radioimmunoassay, and the homeostasis model assessment of insulin resistance index (HOMA-IR) was calculated; the morphological change of pancreas islets was observed by HE staining; the protein expressions of SOCS3 and IRS-1 in hypothalamus were detected by Western blot. Before intervention, compared with the blank group, FPG in the model group and the EA group was increased (P<0.01). After intervention, compared with the blank group, FPG, serum level of FINS and HOMA-IR were increased (P<0.01), the protein expression of SOCS3 was increased while IRS-1 was decreased in the hypothalamus in the model group (P<0.01). Compared with the model group, FPG, serum level of FINS and HOMA-IR were decreased (P<0.01), the protein expression of SOCS3 was decreased while IRS-1 was increased in the hypothalamus in the EA group (P<0.01). In the model group, the shape of pancreas islets was irregular, the area of pancreas islets and the number of islet β cell nuclei were decreased, the nuclei of islet β cell was compensatory enlargement. In the EA group, the shape and the area of pancreas islets and the number of islet β cell nuclei were improved, the compensatory increase of islet β cell nuclei was alleviated compared with the model group. Electroacupuncture can reduce the fasting plasma glucose, improve the morphology of pancreas islets, and alleviate the insulin resistance in ZDF rats. The mechanism may relate to the down-regulation of SOCS3 and up-regulation of IRS-1 in the hypothalamus, and improving the function of hypothalamus in regulating peripheral glucose metabolism.

Thyroid-Stimulating Hormone Inhibits Insulin Receptor Substrate-1 Expression and Tyrosyl Phosphorylation in 3T3-L1 Adipocytes by Increasing NF-κB DNA-Binding Activity.

Background Abundant evidence indicates that thyroid-stimulating hormone (TSH) levels are associated with insulin resistance in adipocytes. However, the potential mechanism of the association remains uncertain. The objective of this study was to determine the potential role of TSH in the suppression of insulin receptor substrate-1 (IRS-1) expression and IRS-1 tyrosyl phosphorylation, which might contribute to insulin resistance. Methods Mouse 3T3-L1 preadipocytes were differentiated into adipocytes. After treatment with 0.01, 0.1, and 1.0 mIU/ml bovine TSH, the TNF-α concentration in the medium was determined by enzyme-linked immunosorbent assay (ELISA). Nuclear factor-kappa B (NF-κB) DNA-binding activity was quantified by electrophoretic mobility shift assay (EMSA). IRS-1 levels in adipocytes were quantified by Western blotting, and tyrosine phosphorylation was measured by immunoprecipitation. Results TSH induced TNF-α secretion in a dose-dependent manner. There was a significant positive correlation between NF-κB DNA-binding activity and TNF-α secretion. This effect and correlation were weakened by BAY 11-7082 (a nuclear NF-κB inhibitor) and H89 (an inhibitor of cyclic adenosine monophosphate- (cAMP-) dependent protein kinase A (PKA)). Treatment of cultured adipocytes with TSH inhibited insulin-stimulated IRS-1 tyrosyl phosphorylation but promoted TSH-dependent secretion of TNF-α and activation of NF-κB DNA-binding activity. The effects of TSH were significantly inhibited by BAY 11-7082 and H89 and were completely blocked by the TNF-α antagonist WP9QY. Conclusion TSH inhibited IRS-1 protein expression and tyrosyl phosphorylation in 3T3-L1 adipocytes by stimulating TNF-α production via promotion of NF-κB DNA-binding activity. TSH might play a pivotal role in the development of insulin resistance.

Changes of LRP6/β-catenin pathway in adipose tissue of rats with intrauterine growth restriction with catch-up growth.

To investigate the expression of low-density lipoprotein receptor-related protein 6 (LRP6)/β-catenin pathway related proteins in adipose tissue of rats with intrauterine growth restriction with catch-up growth SD rats were randomly divided into nutrition-restriction rats and normal feed rats during pregnancy. CG-IUGR model was established by reducing the number of offspring in the nutrition-restriction rats (CG-IUGR group); while the rats in the control group were offspring of the normal feed pregnant rats. In order to exclude the interference of gender, male offspring mice were selected in both the CG-IUGR group and the control group in the following studies. The CG-IUGR group and the control group were subjected to glucose tolerance test at 12 weeks of age, and the perirenal adipose tissue samples were taken to observe the adipose structure by HE staining. Expression of LRP6, β-catenin and insulin receptor substrate 1 (IRS-1) in adipocytes were examined by confocal microscopy. Protein expression of LRP6, β-catenin and IRS-1 were measured by Western blotting. Blood glucose level and the area under the cure of CG-IUGR group were significantly higher than that of control group (both <0.05). Adipocyte size in the CG-IUGR group was significantly larger than that of control group, and the expression of LRP6, β-catenin and IRS-1 protein in adipose tissue of the CG-IUGR group was significantly lower than that of control group (all <0.05). : The expression of LRP6/β-catenin pathway related proteins is reduced in the adipose tissue in CG-IUGR rats, probably contributing to the insulin resistance in these rats.

Magnolia officinalis Ameliorates Dehydroepiandrosterone-Induced Polycystic Ovary Syndrome in Rats

Background: Polycystic ovary syndrome (PCOS) is a prevalent reproductive and metabolic disorder. Insulin resistance (IR) is highly associated with PCOS and aggravates its symptoms. Thiazolidinediones (TZDs), as insulin sensitizing agents, are PPARγ agonists that improve many of the symptoms of PCOS. The Magnolia officinalis extract (MOE) is a natural peroxisome proliferator activated receptor gamma (PPARγ) agonist that improves insulin sensitivity in experimental models. Objectives: Using a dehydroepiandrosterone (DHEA)-induced rat model of PCOS and IR, this study aimed to explore both the potential beneficial effects and the molecular mechanisms of action of MOE. Methods: Post-pubertal female Sprague Dawley rats were subcutaneously injected daily with DHEA (6 mg/100 g body weight) dissolved in sesame oil for 28 days (n = 30). Age- and weight-matched control rats received only sesame oil (n = 12). Afterward, 16 of the DHEA-injected rats, along with five control rats, were sacrificed for blood and tissue collection. The 14 remaining DHEA-injected rats received either treatment of 30 days of oral MOE (500 mg/kg) dissolved in dimethyl sulfoxide (DMSO) (n = 7), or oral DMSO only (n = 7). Meanwhile, the remaining control rats (n = 7) continued to receive daily oral DMSO for 30 days. At the end of the treatments, the rats were sacrificed for blood and tissue collection. Results: After 28 days, the DHEA-treated rats exhibited an increase in body weight as compared to controls (P &lt; 0.05). DHEA injection induced a PCOS phenotype as evident by a statistically significant (P &lt; 0.05) elevated serum luteinizing hormone (LH), and an increased number of cystically dilated follicles with thicker granulosa compared to controls. PCOS rats showed a statistically significant rise in fasting insulin with an increased homeostatic model assessment index of insulin resistance (HOMA-IR) as compared to controls (P &lt; 0.05). Compared to the control group, PCOS rats had a statistically significant lower ovarian protein expression of PPARγ, insulin receptor substrate 1 (IRS1), and protein kinase B (Akt) by Western Blot (P &lt; 0.05). Conversely, the PCOS group showed an increased mammalian target of rapamycin (mTOR) pathway activity as evident by an increase in the fraction of phosphorylated mTOR to total mTOR compared to the control group (P &lt; 0.05). When treated for 30 days with oral MOE (500 mg/kg), the PCOS rats showed a statistically significant decrease in body weight and serum LH levels as compared to the non-treated PCOS rats (P &lt; 0.05). The number of cystically dilated follicles in the MOE-treated PCOS rats was significantly reduced compared to the non-treated PCOS rats. In the MOE-treated PCOS rats, the ovarian protein expression of PPARγ, IRS1, and Akt was significantly increased, while the p-mTOR/mTOR expression was decreased compared to the non-treated PCOS group (P &lt; 0.05). Conclusions: According to our results, the MOE ameliorated the DHEA-induced PCOS phenotype histologically, hormonally, and metabolically. Fundamentally, this explores the elusive pathophysiologic association between IR and PCOS by targeting pathways common to both disorders.

Effects of GPR81 agonist on insulin resistance in rats with nonalcoholic fatty liver disease

Objective: To investigate the effects of NOD-like receptor protein 3 (NLRP3) signaling pathway on insulin resistance and the intervention of lactic acid receptor G protein-coupled receptor 81 (GPR81) agonist in nonalcoholic fatty liver disease (NAFLD) rats. Methods: Thirty SD male rats were randomly divided into three groups: control group, NAFLD group and GPR81 agonist group, with 10 rats in each group. Nonalcoholic fatty liver rat model was established by high fat diet. The rats in GPR81 agonist group were injected intraperitoneally with GPR81 specific agonist lactate (50 nmol/L) on the basis of nonalcoholic fatty liver model once a week, and the other two groups were injected with the same amount of normal saline for 12 weeks. The levels of liver biochemical indexes, fasting blood glucose, insulin and inflammatory factors in liver homogenate were measured, and the histopathological morphology of liver in each group was observed. The protein expressions of NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC), cysteinyl aspartate specific proteinase-1 (caspase-1), insulin receptor substrate 1 (IRS-1), Tyr465-IRS-1, Ser636-IRS-1, glucose transporter 4 (GLUT4) in liver tissue were detected by Western blot. The mRNA expression levels of NLRP3, ASC, caspase-1, IRS-1 and GLUT4 in liver tissue were detected by qRT-PCR. Results: Compared with the control group, the serum levels of triglyceride (TG), alanine aminotransfease (ALT), aspartate aminotransfease (AST), fasting plasma glucose (FPG), fasting insulin (FINS) and homeostasis model assessment of insulin resistance (HOMA-IR) of NAFLD group were increased significantly (P＜ 0.05). The results of liver histopathology showed that in NAFLD group, there were obvious fatty changes in liver tissue, fat droplets in hepatocytes and inflammatory cell infiltration. And the mRNA and protein expressions of NLRP3, ASC, caspase-1 and the protein expression of Ser636-IRS-1 in NAFLD group were increased significantly, and the contents of interleukin 1β (IL-1β) and interleukin 18 (IL-18) in liver and serum were increased, while the mRNA and protein expressions of IRS-1 and GLUT4 and Tyr465-IRS-1 were decreased significantly (P＜0.05). Compared with NAFLD group, the above indexes of GPR81 agonist group were all significantly improved. Conclusion: The activation of NLRP3 signaling pathway mediates the production of inflammatory factors and promotes the development of NAFLD. GPR81 agonist may be a potential treatment for NAFLD.

Downregulation of microRNA‑126 is inversely correlated with insulin receptor substrate‑1 protein expression in colorectal cancer and is associated with advanced stages of disease

Colorectal cancer (CRC) is a common human malignant tumor, and the fourth most common cause of cancer-associated mortality in China. However, the pathogenesis of CRC is not yet fully understood. The present study aimed to investigate the expression and clinical significance of microRNA (miR)-126 and insulin receptor substrate-1 (IRS-1), as well as the role of miR-126 in the prognosis of patients with CRC. A total of 86 colorectal tissue specimens, including 40 CRC and adjacent normal tissue, 26 colorectal adenoma tissue and 20 normal colorectal tissue samples, were collected for the present study. Reverse transcription-quantitative PCR analysis was performed to determine miR-126 and IRS-1 mRNA expression levels, while western blotting and immunohistochemistry (IHC) analyses were performed to determine IRS-1 protein expression levels. The correlation between miR-126 and IRS-1 expression, as well as the association between altered miR-126 and IRS-1 expression levels and clinicopathological characteristics, and the overall survival time of patients with CRC were assessed. The results demonstrated that miR-126 expression was significantly downregulated, while IRS-1 protein expression was upregulated in CRC tissues compared with that in adjacent normal tissues, colorectal adenoma tissues and normal colorectal tissues, respectively. IHC analysis exhibited strong positive staining of IRS-1 protein in CRC tissues, while absent or weak staining of IRS-1 protein was detected in adjacent normal tissues, colorectal adenoma tissues and normal colorectal tissues. miR-126 expression was inversely correlated with IRS-1 protein expression in CRC tissues (r=−0.420; P<0.05). Furthermore, downregulated miR-126 expression was associated with advanced clinicopathological characteristics of the disease and a shorter overall survival time in patients with CRC. Taken together, the results of the present study suggest that miR-126 downregulation may be a candidate molecular marker predictive of poor prognosis of patients with CRC.

The Effect of Magnolol on a Polycystic Ovary Syndrome Rat Model with Insulin Resistance

Polycystic ovary syndrome (PCOS) is a prevalent reproductive and metabolic disorder. Insulin resistance (IR) is highly associated with and aggravates the symptoms of PCOS. The Insulin sensitizing agents, Thiazolidinediones, are PPARγ agonists that improve many of the symptoms of PCOS. Magnolol is a natural PPARγ agonist that improves insulin sensitivity in experimental models. Using a dehydroepiandrosterone (DHEA)‐induced rat model of PCOS and IR, we aim to explore both the potential beneficial effect and the molecular mechanisms of action of Magnolol. Post‐pubertal female Sprague Dawley rats, injected subcutaneously with DHEA (6mg/100g body weight) for 28 days, exhibited a significant increase in body weight (242.5 ± 5.4 grams) as compared to age‐ and weight‐matched controls (226.8 ± 4.7 grams). The DHEA‐injected rats developed an increased number of cystically dilated follicles with thicker granulosa as compared to control. PCOS rats showed statistically significant elevated serum levels of Luteinizing hormone (LH) (10.0 ± 2.7 ng/ml) and fasting insulin (2.18 ± 0.9 ng/ml); with an increased Homeostatic Model Assessment Index of insulin resistance (HOMA‐IR) (9.98 ± 4.9) as compared to controls (6.3 ± 0.2 ng/ml; 0.6 ± 0.3 ng/ml; 2.7 ± 3.9 respectively). Compared to control, PCOS rats had a significant lower ovarian protein expression of peroxisome proliferator activated receptor gamma (PPARγ), insulin receptor substrate 1 (IRS1), and protein kinase B (Akt) by Western Blot. Conversely, the PCOS group showed an increased mammalian target of rapamycin (mTOR) pathway activity as evident by an increase protein expression of phosphorylated mTOR as compared to control. When treated for 28 days with oral Magnolol (500mg/kg), the DHEA‐induced PCOS rats showed a statistically significant decrease in body weight (231.6 ± 13 grams) and serum LH levels (3.9 ± 1.8 ng/ml) as compared to the non‐treated PCOS rats (250.3 ± 9.7 grams; 9.5 ± 5.7 ng/ml respectively). The number of cystically dilated follicles in the Magnolol‐treated PCOS rats was significantly reduced compared to the non‐treated PCOS rats. In the Magnolol‐treated PCOS rats the ovarian protein expression of PPARγ, IRS1 and Akt was significantly increased, while the mTOR expression was decreased compared to the non‐treated PCOS group. In conclusion, Magnolol ameliorates the DHEA‐induced PCOS phenotype histologically, hormonally and metabolically. Fundamentally, this work explores the elusive pathophysiologic association between IR and PCOS, by targeting pathways common to both disorders.Support or Funding InformationThis work is supported by the University of Balamand, Faculty of Medicine and Medical Sciences

Dual-specificity tyrosine phosphorylation-regulated kinase 1A ameliorates insulin resistance in neurons by up-regulating IRS-1 expression

Insulin resistance in the brain is a pathological mechanism that is shared between Alzheimer's disease (AD) and type 2 diabetes mellitus (T2DM). Although aberrant expression and phosphorylation of insulin receptor substrate 1 (IRS-1) contribute to insulin resistance, the underlying mechanism remains elusive. In this study, we used several approaches, including adeno-associated virus-based protein overexpression, immunoblotting, immunoprecipitation, immunohistochemistry, and in situ proximal ligation assays, to investigate the function of dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) in IRS-1 regulation and the downstream insulin signaling in neurons. We found that DYRK1A overexpression up-regulated IRS-1 expression by slowing turnover of the IRS-1 protein. We further observed that DYRK1A directly interacted with IRS-1 and phosphorylated IRS-1's multiple serine residues. Of note, DYRK1A and IRS-1 were coordinately up-regulated in the prefrontal cortex of db/db mice brain. Furthermore, DYRK1A overexpression ameliorated chronic high insulin-induced insulin resistance in SH-SY5Y cells as well as in primary rat neurons. These findings suggest that DYRK1A protects against insulin resistance in the brain by elevating IRS-1 expression.

Yunpi Heluo decoction attenuates insulin resistance by regulating liver miR-29a-3p in Zucker diabetic fatty rats

Background and objectiveYunpiheluo (YPHL) decoction is a Chinese herbal formula with unique advantages for the treatment of type 2 diabetes mellitus (T2DM). The aim of the present study was to investigate changes in miRNA expression and downstream gene expression in Zucker diabetic fatty (ZDF) rats treated with YPHL to determine whether YPHL could be used as an adjuvant treatment of T2DM. MethodsSerum and liver total cholesterol (TC) and triglycerides (TG) levels, insulin resistance index (IR) and differentially expressed miRNAs were detected in a T2DM ZDF rat model. miRNA target prediction was based on bioinformatic algorithms and dual luciferase reporter assay. Protein expression of genes in the insulin receptor signaling pathway was detected by Western blot. The IR cell model was established and the effects of lyophilized YPHL powder on the protein expressions were observed by transfecting specific miRNA mimics and inhibitors. ResultsThe miR-29a-3p expression level was significantly increased in the liver of ZDF rats. Insulin receptor substrate 1 (IRS1) was the target gene of miR-29a-3p. IRS1 mRNA and protein expressions of IRS1, IRS1 (phospho S307), protein kinase B (Akt), Akt (phosphor ser473) and pyruvate dehydrogenase lipoamide kinase isozyme 1 (PDK1) were decreased significantly. miR-29a-3p over-expression decrease IRS1 and the others protein expressions in the HepG2 IR cell model while anti-miR-29a-3p showed the opposite result. The miR-29a-3p level was decreased, and the expressions of IRS1 mRNA and the above proteins were all increased after YPHL treatment. ConclusionmiR-29a-3p played a functional role in insulin receptor signaling in the liver of ZDF rats. YPHL decoction attenuated IR in T2DM probably by down-regulating or maintaining the miR-29a-3p level, increasing the expression of IRS1 mRNA and its phosphorylated proteins, and regulating the expression of insulin receptor signaling-related proteins. YPHL may prove to be an alternative treatment for T2DM.

Hesperidin ameliorates insulin resistance by regulating the IRS1-GLUT2 pathway via TLR4 in HepG2 cells.

The aim of this study was to evaluate the effect and mechanism of hesperidin (HES) on insulin resistance (IR) in the human hepatocellular carcinoma cell line (HepG2 cells). HepG2 cells were induced with lipopolysaccharide (LPS) as a model of IR and treated with HES at three dosages. Next, the levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), the glucose content, and glucose uptake were evaluated by enzyme-linked immunosorbent assay, glucose oxidase-peroxidase method (GOD-POD), or (2-(N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)amino)-2-deoxyglucose) (2-NBDG). Moreover, the protein expression of toll-like receptors 4 (TLR4), insulin receptor substrate 1 (IRS1), nuclear factor kappa B (NF-κB), and glucose transporter 2 (GLUT2) in HepG2 cells treated with HES were assessed via western blotting analysis. In addition, GLUT2 protein expression exposed to HES was detected following treatment with TLR4 inhibitor (HTA125). Our results demonstrated that HES decreased the levels of TNF-α and IL-6, attenuated the glucose content in culture medium and increased glucose uptake in insulin-resistant HepG2 cells in vitro. Moreover, HES upregulated the expression of IRS1 and GLUT2 protein and downregulated the protein expression of TLR4 and NF-κB in insulin-resistant HepG2 cells. The expression of GLUT2 protein had no significant changes when treated with HES after blockade of TLR4. HES attenuated IR in LPS-inducedinsulin-resistant HepG2 cells. Therefore, regulating the IRS1-GLUT2 pathway via TLR4 represents a potential mechanism of HES on IR in HepG2 cells.

Treatment of type 2 diabetes mellitus with the stem and leaf of blacktartary buckwheat and its effects on the pancreas and spleen

To investigate the therapeutic effects of the black buckwheat leaf (BBL) in type 2 diabetes mellitus mice and its effects on pancreas and spleen. Forty male C57 / B16 mice (SPF) were randomly divided into normal control (NC) group (n=10) and the experimental group (n=30), the experimental group were fed with high sugar and high fat, combined with intraperitoneal injection of streptozotocin in small dose to establish the model of type 2 diabetes mellitus (T2DM). Those thirty model mice were randomly divided into 3 groups (n=10), diabetes mellitus group (DM), low dose of BBL (DM+L) treated group, high dose of BBL (DM+H) treated group. The mice in the NC group and the DM group were given normal saline per day, and the DM+L group and DM+H group were treated with black tartary buckwheat at the doses of 0.21g/kg·d-1 and 0.42g/kg·d-1 respectively. After 14 days. All mice were executed by cervical dislocation, then blood samples were collected, pancreas and spleen were removed for subsequent experiments. The serum levels of fasting blood glucose (FBG), triglyceride (TG), total cholesterol (TCH) and insulin were detected. TNF-α protein in spleen tissue was detected by ELISA kit. The morphology of pancreas tissue was observed by HE staining, and the spleen coefficient was calculated. The expression levels of insulin receptor substrate-1(IRS-1) mRNA and IRS-1 protein in pancreatic tissue were detected. Compared with the control group, the serum levels of FBG, TC and TCH in the model group were increased significantly, while the serum level of insulin was decreased significantly (P＜0.05), the expression of TNF-α protein in spleen tissues was obviously raised, the expressions of IRS-1 mRNA and IRS-1 protein in pancreatic tissue in model group were decreased significantly (P＜0.05). Compared with the model group, the serum levels of FBG, TC and TCH were decreased significantly in the BBL treated groups. The serum insulin level, spleen coefficient, TNF-α protein expression level in spleen tissue, IRS-1 mRNA expression and IRS-1 protein expression levels in pancreatic tissue in BBL treated group were increased significantly (P＜ 0.05). High-dose black tartary buckwheat leaves (0.42g/kg·d-1) exerted a more significant effect. Stem and leaf of black bitter buckwheat has significant therapeutic effects on reducing blood sugar and blood fat in type 2 diabetic mice, and has certain protective effects on pancreas and spleen of diabetic mice.

PGBR extract ameliorates TNF-α induced insulin resistance in hepatocytes

Pre-germinated brown rice (PGBR) could ameliorate metabolic syndrome, however, not much research estimates the effect of PGBR extract on insulin resistance. The aim of this study is to examine the effects of PGBR extract in TNF-α induced insulin resistance. HepG2 cells, hepatocytes, were cultured in DMEM medium and added with 5 μM insulin or with insulin and 30 ng/ml TNF-α or with insulin, TNF-α and PGBR extract (50, 100, 300 μg/ml). The glucose levels of the medium were decreased by insulin, demonstrating insulin promoted glucose uptake into cell. However, TNF-α inhibited glucose uptake into cells treated with insulin. Moreover, insulin increased the protein expressions of AMP-activated protein kinase (AMPK), insulin receptor substrate-1 (IRS-1), phosphatidylinositol-3-kinase-α (PI3K-α), serine/threonine kinase PI3K-linked protein kinase B (Akt/PKB), glucose transporter-2 (GLUT-2), glucokinase (GCK), peroxisome proliferator activated receptor-α (PPAR-α) and PPAR-γ. TNF-α activated p65 and MAPKs (JNK1/2 and ERK1/2) which worsened the expressions of AMPK, IRS-1, PI3K-α, Akt/PKB, GLUT-2, GCK, glycogen synthase kinase-3 (GSK-3), PPAR-α and PPAR-γ. Once this relationship was established, we added PGBR extract to cell with insulin and TNF-α. We found glucose levels of medium were lowered and that the protein expressions of AMPK, IRS-1, PI3K-α, Akt/PKB, GLUT-2, GCK, GSK-3, PPAR-α, PPAR-γ and p65, JNK1/2 were also recovered. In conclusion, this study found that TNF-α inhibited insulin stimulated glucose uptake and aggravated related proteins expressions, suggesting that it might cause insulin resistance. PGBR extract was found to ameliorate this TNF-α induced insulin resistance, suggesting that it might be used in the future to help control insulin resistance.

Downregulation of miR-139-5p contributes to the antiapoptotic effect of liraglutide on the diabetic rat pancreas and INS-1 cells by targeting IRS1.

Liraglutide is administered as glucagon-like peptide-1 (GLP-1) receptor agonist for diabetic patients and can protect pancreatic β-cells by inhibiting their apoptosis. MicroRNA-139-5p (miRNA-139-5p) participates in the regulation of cancer cell apoptosis. However, it is not clear whether miR-139-5p contributes to the anti-apoptotic effect of liraglutide in β-cells. The objective of the present study was to investigate the role of miR-139-5p on apoptosis of pancreatic β-cells. MicroRNA levels in pancreatic tissue from diabetic rats and INS-1 cells treated with liraglutide were measured by real-time quantitative RT-PCR. The role of miR-139-5p on apoptosis was studied by transfecting INS-1 cells with miR-139-5p mimics. The mRNA and protein expression of the target gene, insulin receptor substrate-1 (IRS1), were measured by qRT-PCR and Western blot, respectively. Apoptosis in rat pancreatic tissue and INS-1 cells was detected by TUNEL and annexin V/propidium iodide costaining. Apoptosis of pancreatic tissue from diabetic rats and INS-1 cells was decreased by administration of liraglutide. The expression of miR-139-5p increased in the pancreas of diabetic rats and decreased with liraglutide treatment. Incubation with liraglutide (100 nM) for 48 h attenuated the expression of miR-139-5p and increased the mRNA and protein levels of IRS1. Direct regulatory effects of miR-139-5p on IRS1 were found by a dual-luciferase reporter assay. Transfection of INS-1 cells with miR-139-5p mimics led to decreases in the mRNA and protein expression of IRS1. In conclusion, our observations suggest that decreased miR-139-5p expression contributes to the anti-apoptotic effect of liraglutide on the diabetic rat pancreas and INS-1 cells by targeting IRS1.

IRS1/β-Catenin Axis Is Activated and Induces MYC Expression in Acute Lymphoblastic Leukemia Cells.

Insulin-like growth factor 1 (IGF1) and its receptor IGF1R regulate normal cell growth and contribute to cell transformation through activation of downstream signaling pathways. In fibroblast cells, insulin receptor substrate 1 (IRS1), through IGF1 signaling, was found to be the key protein for nuclear translocation of β-catenin and MYC transcription activation. We herein investigated the IRS1/β-catenin axis in acute lymphoblastic leukemia (ALL) cells. Samples were obtained from 45 patients with ALL and 13 healthy donors. ALL cell lines were used. Gene expression was measured by quantitative PCR. Protein expression, associations, and cellular localization were evaluated by immunoprecipitation, subcellular fractionation, and confocal microscopy. Cells were submitted to IGF1 stimulation and/or IGF1R pharmacological inhibition (OSI-906). IRS1, β-catenin, and MYC mRNA expression were significantly elevated in ALL patients, compared to normal controls. MYC mRNA expression positively correlated with β-catenin and IRS1. Increased age and MYC expression negatively affected overall survival by univariate analysis. Total and phospho-IGF1R and IRS1, MYC and β-catenin protein expression were higher in ALL cells, compared to normal peripheral blood mononuclear cells (PBMC). IRS1 and β-catenin were found to be colocalized in the nuclei and the cytoplasm of ALL cell lines, whereas both proteins were only slightly detected in the cytoplasm of normal PBMC. In Jurkat cells, a constitutive IRS1 and β-catenin protein interaction were observed; OSI-906 treatment decreased IGF1R tyrosine phosphorylation, IRS1 expression and phosphorylation, nuclear translocation of β-catenin, IRS1 and β-catenin association, and MYC protein expression. In conclusion, the IRS1/β-catenin axis is activated in ALL cells. J. Cell. Biochem. 118: 1774-1781, 2017. © 2016 Wiley Periodicals, Inc.

MiR-195 inhibits tumor growth and angiogenesis through modulating IRS1 in breast cancer.

Angiogenesis has been found as an attractive target for drug therapy as it is necessary for tumor growth. Accumulating evidences show that microRNAs (miRNAs), which are a group of highly conserved, single-stranded, short non-coding RNAs, play important roles through directly targeting angiogenic factors and protein kinases. The purpose of this study is to investigate the role of miR-195 in breast cancer development and angiogenesis through targeting IRS1. We show that miR-195 is inversely related with Insulin receptor substrate 1 (IRS1) in both breast cancer cells and breast cancer tissues. Induction of miR-195 could suppress IRS1 protein expression through binding to its 3'UTR regions either by transfection with miR-195 oligo or by infection with lentivirus encoding miR-195 gene. Moreover, re-expression of IRS1 reverses miR-195-mediated repression of tumor cell growth and miR-195 inhibits tumor angiogenesis through suppressing IRS1-VEGF axis. These data suggest that miR-195 mimics are potential therapeutic agents for breast cancer diagnose.

MiR-628 Promotes Burn-Induced Skeletal Muscle Atrophy via Targeting IRS1.

Skeletal muscle atrophy is a common clinical feature among patients with severe burns. Previous studies have shown that miRNAs play critical roles in the regulation of stress-induced skeletal muscle atrophy. Our previous study showed that burn-induced skeletal muscle atrophy is mediated by miR-628. In this study, compared with sham rats, rats subjected to burn injury exhibited skeletal muscle atrophy, as well as significantly decreased insulin receptor substrate 1 (IRS1) protein expression and significantly increased skeletal muscle cell apoptosis. An miRNA array showed that the levels of miR-628, a potential regulator of IRS1 protein translation, were also clearly elevated. Second, L6 myocyte cell apoptosis increased after induction of miR-628 expression, and IRS1 and p-Akt protein expression decreased significantly. Expression of the cell apoptosis-related proteins FoxO3a and cleaved caspase 3 also increased after induction of miR-628 expression. Finally, forced miR-628 expression in normal rats resulted in increased cell apoptosis and skeletal muscle atrophy, as well as changes in IRS1/Akt/FoxO3a signaling pathway activity consistent with the changes in protein expression described above. Inhibiting cell apoptosis with Z-VAD-FMK resulted in alleviation of burn-induced skeletal muscle atrophy. In general, our results indicate that miR-628 mediates burn-induced skeletal muscle atrophy by regulating the IRS1/Akt/FoxO3a signaling pathway.

Urotensin II-induced insulin resistance is mediated by NADPH oxidase-derived reactive oxygen species in HepG2 cells.

To investigated the effects of urotensin II (UII) on hepatic insulin resistance in HepG2 cells and the potential mechanisms involved. Human hepatoma HepG2 cells were cultured with or without exogenous UII for 24 h, in the presence or absence of 100 nmol/L insulin for the last 30 min. Glucose levels were detected by the glucose-oxidase method and glycogen synthesis was analyzed by glycogen colorimetric/fluorometric assay. Reactive oxygen species (ROS) levels were detected with a multimode reader using a 2',7'-dichlorofluorescein diacetate probe. The protein expression and phosphorylation levels of c-Jun N-terminal kinase (JNK), insulin signal essential molecules such as insulin receptor substrate -1 (IRS-1), protein kinase B (Akt), glycogen synthase kinase-3β (GSK-3β), and glucose transporter-2 (Glut 2), and NADPH oxidase subunits such as gp91(phox), p67(phox), p47(phox), p40(phox), and p22(phox) were evaluated by Western blot. Exposure to 100 nmol/L UII reduced the insulin-induced glucose consumption (P < 0.05) and glycogen content (P < 0.01) in HepG2 cells compared with cells without UII. UII also abolished insulin-stimulated protein expression (P < 0.01) and phosphorylation of IRS-1 (P < 0.05), associated with down-regulation of Akt (P < 0.05) and GSK-3β (P < 0.05) phosphorylation levels, and the expression of Glut 2 (P < 0.001), indicating an insulin-resistance state in HepG2 cells. Furthermore, UII enhanced the phosphorylation of JNK (P < 0.05), while the activity of JNK, insulin signaling, such as total protein of IRS-1 (P < 0.001), phosphorylation of IRS-1 (P < 0.001) and GSK-3β (P < 0.05), and glycogen synthesis (P < 0.001) could be reversed by pretreatment with the JNK inhibitor SP600125. Besides, UII markedly improved ROS generation (P < 0.05) and NADPH oxidase subunit expression (P < 0.05). However, the antioxidant/NADPH oxidase inhibitor apocynin could decrease UII-induced ROS production (P < 0.05), JNK phosphorylation (P < 0.05), and insulin resistance (P < 0.05) in HepG2 cells. UII induces insulin resistance, and this can be reversed by JNK inhibitor SP600125 and antioxidant/NADPH oxidase inhibitor apocynin targeting the insulin signaling pathway in HepG2 cells.